Aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) herbicides are used post-emergence in dicot crops to control gramineous weeds. Because these herbicides effectively kill most monocotyledonous species at low concentrations, there is low toxicity to non-target organisms. Great potential exists for developing cereal varieties that can be treated post-emergence to control weedy grasses that escape other pre-emergent herbicides treatments (Somers, 1996). Furthermore, these herbicides have low persistence in soil and provide growers with increased flexibility for weed control and crop rotation. For example, red rice is the most pervasive and expensive pest in U.S. rice production (USDA-ARS Dale Bumpers National Rice Research Center 2006 Annual Report) and can serve as a host for rice diseases. CLEARFIELD® Rice is the premier tool for managing red rice in infested areas; however, gene flow between red rice and CLEARFIELD® Rice can result in ˜170 FI hybrids/ha (Shivrain et al., 2007). Thus, stewardship guidelines limit CLEARFIELD® Rice market penetration to two out of any four years on any given field. Therefore, the generation of cultivated rice with tolerance to different herbicides will provide farmers CLEARFIELD® with a rotation partner to help manage red rice weed populations.
FOPs and DIMs target the enzyme Acetyl-CoA Carboxylase (EC 6.4.1.2), which catalyzes the first committed step in fatty acid (FA) biosynthesis. ACCase is a biotinylated enzyme that converts acetyl-CoA to malonyl-CoA in a 2-step reversible reaction. The enzyme first carboxylates the biotin group and then the intrinsic carboxytranferase activity transfers the carboxyl group from carboxybiotin to acetyl-CoA (Nikolau et al., 2003). ACCase activity is necessary in the plastid which is the primary site for FA biosynthesis for membrane biogenesis. ACCase activity is also present in the cytosol, where it is involved in the synthesis of very long chain FA and flavonoids (Chugh and Eudes, 2008). The multidomain, cytoplasmic monocot and dicot ACCases are not sensitive to the FOP and DIM herbicides. It is the plastidic form of ACCase that confers the selectivity to this class of herbicides. Dicot plastidic ACCases are naturally insensitive while monocot plastidic ACCases are herbicide-sensitive. The plastidic ACCases are highly expressed in meristematic tissue to feed the high demand for membrane biogenesis in rapidly growing, young seedlings (Podkowinski et al., 2003). This essential role for growth explains the effectiveness of targeting ACCase inhibition in post-emergent weeds.
Since the inception of FOP and DIM use for controlling weeds in world agriculture in the 1980s, there has been an emergence of tolerance amid various weed species. Among these, the most extensively studied are Alopecurus myosuroides (blackgrass) and Avena sterilis (wild oat). Comprehensive studies of natural blackgrass and wild oat mutants have revealed six residues within the carboxyltransferase domain of the plastidic isoform that confer tolerance to FOPs and/or DIMs (Delye et al., 2003; Delye et al., 2005; Liu et al., 2007) and these are I1781L, W2027C, I2041N, G2096A, D2078G and C2088R (designation according to standard blackgrass ACCase reference sequence). Interestingly, I1781L and D2078G confer tolerance to both FOPs and DIMs while the other four mutations confer tolerance only to FOPs, suggesting that the binding sites of the two classes of herbicides is overlapping, yet distinct.
Two approaches to develop DIM tolerant rice that have been tried include the following. In the first approach, previously identified mutations in natural blackgrass and wild oat are introduced by means of molecular biological techniques in rice plastidic ACCase. The effects of the mutations are studied in rice after Agrobacterium-mediated transformation of the modified ACCase genes. Ultimately, the same mutations can be engineered at the endogenous locus through oligonucleotide gene targeting (Beetham et al., 1999). In the second approach rice callus is propagated in medium with gradually increasing DIM concentrations. This procedure can enrich the callus for cells in which plastidic ACCase has mutated to become more tolerant to the herbicide. Plants can be regenerated from the callus when a satisfactory tolerance level has been reached. However, both approaches are limited in the number of mutations that can be generated and/or tested.
Yeast is an excellent model organism for screening and testing large numbers of mutated rice ACCase genes for increased herbicide tolerance. However, yeast contains a single, endogenous ACCase gene (ACC1), which encodes a multidomain protein that is highly tolerant to herbicides. Haploid yeast in which ACC1 is disrupted is not viable (Hasslacher et al., 1993). Joachimiak et al. (1997) introduced wheat cytoplasmic ACCase into a diploid strain heterozygous for ACC1. Standard tetrad analysis demonstrated that the ACC1 mutation was complemented by the plant ACCase. A similar experiment with the herbicide-sensitive wheat plastidic ACCase gene showed that this gene was not able to take over the function of ACC1 (Nikolskaya et al., 1999). A series of chimeric constructs consisting of the N-terminus of wheat cytoplasmic ACCase and the C-terminus of wheat plastidic ACCase was tested for complementation and herbicide sensitivity. Wheat c60p40 ACCase, which is comprised of the first 60% of cytoplasmic ACCase and last 40% of plastidic ACCase, complemented an ACCase deletion mutant, Δacc1, while showing the highest sensitivity towards haloxyfop, clodinafop, quizalofop, cethoxydim and sethoxydim of all constructs tested. The c60p40 chimeric ACCase is a suitable target for mutagenesis, as all herbicide-conferring mutations known have been mapped to the last 40% of plastidic ACCase. Yet, the Joachimiak approach is limited in its efficiency in that each new mutant plastid ACCase construct is separately introduced into yeast cells to create a new yeast knockout complement.
DIMs and FOPs are important herbicides and there is a need for methods and compositions to isolate, identify and characterize herbicide tolerant ACCase variants. The methods and compositions described herein are suitable for isolating, identifying, and characterizing such herbicide tolerant ACCase variants. Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.